Scanning isoelectric focusing in small density-gradient columns. IV. The use of deuterium oxide for preparing the density gradient and its effects on isoelectric points of proteins.

The use of deuterium oxide as a substitute for sucrose in preparing density gradients for small isoelectrofocusing columns has been investigated. A density gradient was created directly in a 1.5-ml column by free inter-diffusion of three deuterium oxide solutions for 3 min. The resulting deuterium oxide concentration course (as shown by measurement of the refractive-index gradient) had a very high degree of linearity. Test runs with beta-lactoglobulin and sperm-whale myoglobin showed that the strength and stability of the deuterium oxide density gradient should normally be sufficient for stabilization of protein zones against convection during isoelectric focusing. The isoelectric points of beta-lactoglobulins A and B were found to increase as the concentration ratio of deuterium oxide to water at focusing level increased. Within the limits of experimental error, the pI shift corresponding to a given change in solvent composition was equal for both components. These findings are accounted for in terms of the deuterium isotope effect on the dissociation constants of protolytic groups and the shift in the asymmetry potential of the glass electrode in deuterium oxide as compared with water. The spontaneous reduction of sperm-whale metmyoglobin to ferrous myoglobin, which occurs on prolonged isoelectric focusing, is discussed, as are the benefits and drawbacks of deuterium oxide as a density-gradient solute.